During the erythrocytic cell cycle, the malaria parasite undergoes three morphologically distinct transitions. It matures from the small ring into the trophozite form, which in turn matures into a segmented schizont. Maturation of the parasite to trophozoite and schizont stages leads to dramatic changes in erythrocyte shape and deformability. The mature parasite finally ruptures the erythrocyte membrane to facilitate the release of progeny merozoites which reinvade new red blood cells. It is known that an extensive modification of the erythrocyte membrane takes place by parasite-derived molecules during the last stages of parasite development. The focus of this proposal is to elucidate mechanisms by which the malaria parasite modifies the constituents of the erythrocyte membrane skeleton during parasite development to ensure its exit from host erythrocytes. Experimentally, our focus will be to explore our recent observation that a 80 kDa erythrocyte membrane protein becomes specifically phosphorylated in trophozoite-containing human red cells. Peptide mapping analysis indicates that the 80 kDa phosphoprotein is structurally similar to host protein 4.1 and its phosphorylation is mediated via a novel, parasite-derived, casein kinase. We will test the hypothesis that phosphorylation of protein 4.1 by the parasite casein kinase abolishes spectrin-actin crosslinking activity of protein 4.1 in vitro. Furthermore, we propose to determine the primary sequence of the phosphorylation sites on the 80 kDa protein and to isolate and characterize the parasite casein kinase. Our anticipation is that the results of these experiments will be helpful to design inhibitors against parasite kinase which may block parasite development and release in vivo. Recently it was shown that antibodies against MESA specifically immunoprecipitated a phosphorylated form of protein 4.1. MESA is a parasite-derived phosphoprotein which associates with the erythrocyte membrane skeleton. Our aim is to examine whether the 80 kDa protein phosphorylated in mature-parasite infected erythrocytes associates with MESA. We will also examine the effect of MESA-protein 4.1 Interaction on the spectrin-actin crosslinking activity of protein 4.1 and the substrate specificity of protein 4.1 to parasite casein kinase. Moreover, we will extend our recent observation that erythrocyte membrane ankyrin and protein 4.1 are specifically degraded by a protease fraction derived from mature parasites. We will: (a) Test whether ankyrin and protein 4.1 are degraded in intact erythrocytes as a function of intracellular parasite development prior to host membrane rupture. (b) Examine whether proteolysis of ankyrin and protein 4.1 affect their binding activities in vitro. (c) Isolate and characterize ankyrin/protein 4.1 specific parasite protease(s) with the expectation that specific inhibitors against parasite protease(s) may block intracellular parasite development and its release in vivo. In summary, the focus of this proposal is to delineate molecular interactions of the human erythrocyte membrane skeleton altered by the developing malaria parasite. Such alterations may likely lead to membrane 'weakening' and its subsequent rupture. Indeed it is the rupture of the erythrocyte membrane and release of progeny merozoites which account for the devastating clinical symptoms of malaria.